Early-life experiential and environmental conditions, particularly those occurring during heightened periods of brain plasticity, are known to promote long-term changes in physiology and behavior that act independently of changes in the DNA code. Accumulating evidence suggests an important role for epigenomic processes in these epigenetic phenomena. In particular, recent data from a variety of sources suggest that experience-dependent changes in DNA methylation can have a long-lasting impact on neural function through sustained effects on neuronal gene expression. However, the fact that these modifications occur in vivo in a relatively small number of cells within highly heterogeneous neural tissue limits the study of these modifications with existing genomic approaches and greatly complicates investigation of the underlying molecular mechanisms. We propose a two-pronged approach to begin to address these issues. First, we will pursue a reductionist approach to the study of experience-driven changes in DNA methylation, employing a dissociated neuronal culture system that shows activity-induced changes in DNA methylation and in which a large number of cells can be synchronously activated with robust stimuli. Moreover, to complement and address limitations inherent in this reductionist approach, we have also developed a general genetic strategy to specifically isolate chromatin from defined cell types in vivo, enabling the analysis of DNA methylation changes induced in specific neuronal cell populations in response to early-life experiences using massively parallel sequencing techniques. Thus, we propose: 1) To employ a dissociated neuronal culture system to characterize neuronal activity-induced changes in DNA methylation, and 2) To investigate long-lasting neuronal epigenomic correlates to experience-driven behavioral and physiological changes in vivo. It is our hope that the proposed experiments will establish new approaches for the analysis of neuronal epigenomic modifications, advance our understanding of the regulation of DNA methylation in the developing central nervous system, and ultimately provide new insights into the importance of these mechanisms for neurodevelopment, cognitive behavior, and disease. PUBLIC HEALTH RELEVANCE: Adverse early life events are known to influence risk for neurodevelopmental and psychiatric disorders, triggering long-lasting changes in physiology and behavior that act independent of changes to the DNA code. In an effort to gain insight into the underlying molecular basis of these effects, the proposed study will explore the role of regulated DNA methylation in the persistent alteration of neuronal gene expression.